While many pain syndromes are thought to have a vascular component, the underlying mechanisms remain elusive. Previous studies have provided strong evidence for a role of mediators that can be released by endothelial cells which line the lumen of blood vessels, but mechanisms by which endothelial cells themselves could dynamically participate in pain generation have not been described. We recently discovered a novel mechanism which drives an active contribution of vascular endothelial cells to peripheral hyperalgesia. This phenomenon, referred to as stimulus-dependent hyperalgesia (SDH) can be elicited by two vasoactive compounds, endothelin-1 (ET-1) and epinephrine, acting at their cognate receptors on the endothelial cell to produce a state in which mechanical stimulation now produces release of ATP that, in turn, acts on P2X3 receptors on sensory neurons. This discovery was made possible by our innovative adaptation of two independent methods from the cardiovascular and renal vascular literature to attenuate endothelial cell function at the site of nociceptive testing: treatment with octoxynol-9 and methionine-induced hyperhomocysteinemia. This grant application outlines experiments to investigate the cellular mechanisms of SDH and to explore the potential role of this or other endothelial cell mechanisms in vascular pain syndromes. In the first Specific Aim, we will investigate mechanisms of SDH including: 1) the role of the endothelial cell in SDH, 2) endothelial cell mediators involved in SDH and mechanisms by which they are released from the endothelial cell, 3) second messenger pathways in endothelial cells involved in SDH, and 4) which nociceptors mediate SDH. In contrast to Specific Aim 1 which probes cellular mechanisms by which vascular endothelial cells actively participate in particular mechanism of SDH, the second Specific Aim will more generally examine the role of the endothelial cell in models of clinical pai syndromes with a vascular component, not limited to the SDH mechanism. This aim will also evaluate the endothelial cell as a possible target of existing treatments for vascular pain syndromes (e.g., triptans and -blockers) the mechanisms of which are not fully understood. Furthermore, since stress is a major factor in clinical pain conditions in which blood vessels are thought to play a role, we will study the impact of neuroendocrine stress axis mediators on endothelialdependent pain in such syndromes. By elucidating the endothelial cell contribution to pain, these studies have the potential to identify novel targets for the development of pharmacological treatments of vascular pain syndromes.